1. Field of the Invention
This invention is in the field of molecular biology and in particular relates to the identification of a novel human Frizzled Related Protein (FRP) involved in cell growth and differentiation.
2. Description of Related Art
Extracellular signaling molecules have essential roles as inducers of cellular proliferation, migration, differentiation, and tissue morphogenesis during normal development. These molecules also participate in many of the aberrant growth regulatory pathways associated with neoplasia. In addition, these molecules function as regulators of apoptosis, the programmed cell death that plays a significant role in normal development and functioning of multicellular organisms, and when disregulated, is involved in the pathogenesis of numerous diseases. See e.g. Thompson, C. B., Science 267, 1456-1462 (1995).
Apoptosis is a result of an active cell response to physiological or damaging agents and numerous gene products are involved in signal transduction, triggering and executive steps of the apoptotic pathways. Other proteins do not take part in the apoptotic cascade by themselves but modify cell sensitivity to proapoptotic stimuli. While many genes and gene families that participate in different stages of apoptosis have recently been identified and cloned, because the apoptotic pathways have not been clearly delineated, many novel genes which are involved in these processes await discovery.
The identification and characterization of molecules involved in growth and differentiation is an important step in both the identification of mechanisms of cellular development and oncogenesis and the subsequent conception of novel therapies based on this knowledge. One group of molecules known to play a significant role in regulating cellular development is the Wnt family of glycoproteins. In vertebrates, this family consists of more than a dozen structurally related molecules, containing 350-380 amino acid residues of which  greater than 100 are conserved, including 23-24 cysteine residues. See e.g. Parr, B. A. and McMahon, A. P. (1994) Curr Opin Genet Dev 4, 523-8.
Wnt-1, the first Wnt-encoding gene to be isolated, was identified as an oncogene expressed as a result of insertional activation by the mouse mammary tumor virus (Nusse, R., et al., Nature 307, 131-6 1984). Subsequently, transgenic expression of Wnt-1 confirmed that constitutive expression of this gene caused mammary hyperplasia and adenocarcinoma (Tsukamoto et. al., Cell 55, 619-25 (1988)). Targeted disruption of the Wnt-1 gene revealed an essential role in development, as mouse embryos had severe defects in their midbrain and cerebellum. Thomas et. al., Cell 67, 969-76 (1991). In addition, Wingless (Wg), the Drosophila homolog of Wnt-1, was independently identified as a segment polarity gene (Rijsewijk et al., Cell 50, 649-57 (1987)). Gene targeting of other Wnt genes demonstrated additional important roles for these molecules in kidney tubulogenesis and limb bud development. See e.g. Parr et al., Nature 374, 350-3 (1995); Stark K et al. Nature 372: 679-683, 1994.
Several aspects of Wnt signaling have been illuminated by studies in flies, worms, frogs and mice (Perrimon, N. (1996) Cell 86, 513-6; Miller, J. R. and Moon, R. T. (1996) Genes Dev 10, 2527-39), but until recently little was known about key events which occur at the external cell surface. Identification of Wnt receptors was hampered by the relative insolubility of the Wnt proteins, which tend to remain tightly bound to cells or extracellular matrix. However, several observations now indicate that members of the Frizzled (FZ) family of molecules including Frzb can function as receptors for Wnt proteins or as components of a Wnt receptor complex. See e.g. He et. al., Science 275, 1652-1654 (1997).
The prototype for this family of receptor molecules, Drosophila frizzled (Dfz), was first identified as a tissue polarity gene that governs orientation of epidermal bristles. Vinson et al., Nature 329, 549-51 (1987). Cells programmed to express a second Drosophila Fz gene, Fz2, bind Wg and transduce a Wg signal to downstream components of the signaling pathway. Bhanot et al., Nature 382, 225-30 (1996). Each member of the Fz receptor gene family encodes an integral membrane protein with a large extracellular portion, seven putative transmembrane domains, and a cytoplasmic tail. See e.g. Wang et al., J Biol Chem 271, 4468-76 (1997). Near the NH2-terminus of the extracellular portion is a cysteine-rich domain (CRD) that is well conserved among other members of the FZ family. The CRD, comprised of xcx9c110 amino acid residues, including 10 invariant cysteines, is the putative binding site for Wnt ligands. Bhanot et al., Nature 382, 225-30 (1996).
In organisms including frogs, fruit flies, and mice, proteins including Wingless, Armadillo, and Frizzled form part of a signaling cascade that controls crucial events during early embryonic developmentxe2x80x94particularly gastrulation, the process by which a hollow ball of embryonic cells collapses in on itself, forming the major embryonic tissues. In vertebrates, the signaling pathwayxe2x80x94headed by the Wnt family of growth factors contribute to the formation of body axis and the proper development of the central nervous system, kidneys, and limbs. When it is activated inappropriately in adult cells, the pathway can precipitate the formation of tumors. During gastrulation, Fz family members may interact with Wnt to control the proper development of the nervous system and muscles. The coupling of Wnt and Frizzled activates a pathway that leads to the expression of a set of Wnt-responsive genes, including those that encode the transcription factors such as Engrailed and Siamois.
When Wnt mRNA is injected into Xenopus embryos in the 4-8 cell stage, the tadpoles develop a second body axis: They can duplicate all or part of the nervous systems from head to tail, and many of their organs are duplicated. Interestingly, during gastrulation, a Wnt family member known as Xwnt-8 serves to xe2x80x9cventralizexe2x80x9d the embryoxe2x80x94steering cells in the mesoderm toward forming muscle. Injecting Frzb mRNA into a developing Xenopus embryo prior to gastrulation inhibits muscle formation, generating tadpoles that are stunted in appearance, with shortened trunks due to the lack of muscle tissue. The embryos also have enlarged heads, because an abnormal number of mesodermal cells adopt a dorsal fate. Knockout mice have already helped researchers understand a few of the various roles that Wnts play in development. To date, scientists have identified 16 different Wnts that function in vertebrate development. Many Wnts appear to be involved in directing the development of the central nervous system (CNS). Others control the formation of nephrons in the kidney and the proper development of the limbs.
The existence of molecules that have a FZ CRD but lack the seven transmembrane motif and cytoplasmic tail suggests that there is a subfamily of proteins that function as regulators of Wnt activity. Little is known about the activity of SDF5, which was cloned using the signal sequence trap method. FRZB is a heparin-binding molecule thought to be involved in skeletal morphogenesis. Recently Rattner et al. cloned cDNAs encoding the murine homologs of Fz family members, and showed that, when artificially linked to the plasma membrane via a glycolipid anchor, SDF5 and FRZB conferred cellular binding to Wg. Rattner et al., P.N.A.S. 94, 2859-2863 (1997).
The disregulation of Wnt pathways appears to be a factor in aberrant growth and development. Mutations in xcex2-catenin, a protein that accumulates when the Wnt pathway is activated, are associated with tumor development in human colon cancers and melanomas. xcex2-catenins couple with other cellular transcription factors and help to activate Wnt-responsive genes. These results confirm that the Wnt-signaling pathway can play an important role in the embryo and the adult. Ultimately, Wnt transmits its signal by allowing xcex2-catenin to accumulate in the cell cytoplasm. There, xcex2-catenin binds to members of the Tcf-Lef transcription factor family and translocates to the nucleus. When Wnt is absent, xcex2-catenin instead forms a complex with glycogen synthase kinase-3 (GSK-3) and the adenomatous polyposis coli (APC) tumorsuppressor protein. This interaction is associated with the phosphorylation of xcex2-catenin, marking it for ubiquitination and degradation. Wnt permits the accumulation of xcex2-catenin by inhibiting the function of GSK-3. The mutations that drive tumor formation follow a similar strategy. Mutations in APC render the tumor-suppressor protein unable to bind to xcex2-catenin, which remains unphosphorylated and accumulates in the cell, turning on Wnt-responsive genes.
Given the potential complexity of interactions between the multiple members of Wnt and FZ families, additional mechanisms might exist to modulate Wnt signaling during specific periods of development or in certain tissues. What is needed in the art is the identification and characterization of novel effectors of the processes which are related to cellular growth and development. The identification of such mechanisms and in particular, the effectors of these mechanisms is important for understanding and modulating the processes of cellular regulation.
The invention includes nucleotide sequences that encode a novel polypeptide, designated in the present application as xe2x80x9cFRPxe2x80x9d (Frizzled Related Protein), which is a secreted antagonist of the Wnt signaling pathway and exhibits a number of characteristics which make it a useful tool for studying cell growth and differentiation as well as oncogenesis. As such, this novel protein has a variety of applications in the identification, characterization and regulation of activities associated with cellular function as well as processes associated with oncogenesis.
The invention provides xe2x80x9cFRPxe2x80x9d (Frizzled Related Protein) polypeptides [SEQ ID Nos. 3,4] and fragments thereof and polynucleotide sequences encoding FRP polypeptides [SEQ ID Nos. 1, 2, and 27]. The invention further provides antibodies which are specific for FRP polypeptides and animals having FRP transgenes. Moreover, the invention provides methods of producing FRP polypeptides and polynucleotide sequences. In addition, the invention provides methods of assaying for FRP in a sample as well as methods for detecting FRP binding partners.
In one embodiment, the invention provides isolated nucleic acid molecules which encode FRP polypeptides. For example, the isolated nucleic acid can include DNA encoding FRP polypeptide having amino acid residues 1 to 313 of FIG. 1 [SEQ ID No. 2, bases 303-1241], or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another embodiment, the invention provides a vector comprising a gene encoding a FRP polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be E. coli, yeast or mammalian cells. A process for producing FRP polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of FRP. If desired, the FRP may be recovered.
In another embodiment, the invention provides isolated FRP polypeptide. In one embodiment, FRP of the invention comprises 313 amino acids and includes a signal sequence, Wnt binding domain, a hyaluronic acid binding domain and potential asparagine-linked glycosylation sites. In particular, the invention provides isolated native sequence FRP polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 313 of FIG. 1 [SEQ ID No. 4]. In a related embodiment, the invention provides chimeric molecules comprising FRP polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule is a factor which includes a FRP fused to a polyhistidine polypeptide sequence. In another embodiment, the invention provides a non-human transgenic animal whose somatic and germ cells contain a transgene comprising human FRP. In yet another embodiment, the invention provides a polypeptide capable of specifically binding a FRP polypeptide such as an antibody specific for a FRP polypeptide. Optionally, the antibody is a monoclonal antibody.
Also included in the invention is a method for regulating cell signaling pathways by inhibiting the interaction of Wnt with Fz receptors by blocking this interaction with FRP molecules. The invention also provides methods for determining the presence of FRP molecules in a sample. The invention also provides a method for determining the presence of Wnt molecules in a sample by screening the sample with FRP. In addition, the invention provides a method for monitoring the course of a neoplastic condition by quantitatively determining the presence of Wnt molecules in a sample by screening the sample with FRP.
In other embodiments, the invention provides methods for using FRP polypeptides and nucleic acids for studying and modulating mechanisms involved in cellular proliferation. In one embodiment, the invention provides a method of modulating cellular phenotype by controlling the level of FRP expression within the cell. In a more specific embodiment, the invention provides a method of inhibiting cellular proliferation and/or differentiation by exposing a cell to FRP.